On the evolution of lignin biosynthesis in plants

木质素是所有维管束植物的一个重要特征。这种酚类聚合物被嵌入木质次生细胞壁中，以提供垂直生长和长距离水分运输所必需的机械稳定性和疏水性。尽管在所有维管植物谱系中普遍存在，但不同的植物谱系中木质素的生物合成途径似乎是不同的。在开花植物中，对大多数木质素单体的限速步骤是由酶4-香豆酰基-莽草酸-3‘-羟基酶(C3’H/CyP98)催化的。然而，在番茄类这种早期分化的维管植物谱系中，向相同木质素单体的生物合成通过不同的途径进行。与之相适应的是，我们最近发现在番茄类中，CYP98不能代谢4-香豆酰-莽草酸，但利用不同的底物，可能从同一途径产生与防御相关的次生代谢物。令人惊讶的是，蕨类植物CYP98也不能使用4-香豆酰-莽草酸，因此蕨类植物中单醇的生物合成似乎也使用了不同的中间体。即使在针叶树中，CYP98底物轮廓也不同于木质素相关的C3‘H酶。因此，我们假设，在陆地植物进化过程中，木质素的生物合成被显着独立地多次修饰。我们将通过对针叶树和蕨类植物中的单个CYP98进行功能表征来检验这一假设。除了详细的生化特征外，我们还需要通过对大规模基因表达数据的分析，将这些CYP98基因的基因表达谱与已知参与木质素生物合成的基因进行比较。还将挖掘这些基因，以确定参与这些谱系中木质素生物合成的新候选基因。重要的是，我们需要建立在这些非模式物种中产生突变的方法，以从功能上测试这些基因在木材形成中的直接参与。我们将产生功能丧失的突变体，这些突变体将表征开花植物cyp98突变体中常见的变化，包括木质素组成的变化。将评估叶片形态和代谢物成分的变化，因为这些变化在非木质素相关的苔藓cyp98突变体中受到影响。这些实验将表明，CYP98是否参与了蕨类和针叶树木质素的生物合成，或者是否已经进化出了其他途径。如果后者成立，从基因表达数据中确定的候选基因将被测试它们的能力，以挽救模式植物拟南芥中cyp98a3功能丧失的表型，如果有必要，然后通过生化和反向遗传方法进一步表征。综上所述，该计划将测试木质素生物合成的进化轨迹，并可能识别对植物征服陆地至关重要的生化途径的进化优化，这条生化途径定义了木材的性质及其人类的用途。

Lignin is a defining feature of all vascular plants. This phenolic polymer is incrusted into woody secondary cell walls to provide mechanical stability and hydrophobicity necessary for upright growth and long-distance water transport. Despite common occurrence in all vascular plant lineages, the biosynthetic route to lignin appears to be different in different plant lineages. In flowering plants, the rate limiting step towards the majority of lignin monomers is catalyzed by the enzyme 4-coumaroyl-shikimate-3'-hydroxylase (C3'H / CYP98). However, in lycopods, an early diverging vascular plant lineage, biosynthesis to the same lignin monomers proceeds through a different route. Fittingly, we have recently shown that in lycopods CYP98 is unable to metabolize 4-coumaroyl-shikimate, but utilizes different substrates, presumably to produce defense related secondary metabolites derived from the same pathway. Surprisingly, a fern CYP98 was also unable to use 4-coumaroyl-shikimate, and thus monolignol biosynthesis in ferns also appears to employ distinct intermediates. Even in conifers, CYP98 substrate profiles are distinct from lignin related C3'H enzymes. We thus hypothesize that lignin biosynthesis was modified significantly independently multiple times during land plant evolution. We will test this hypothesis by functionally characterizing the single CYP98s from a conifer and from a fern species. Besides detailing biochemical characterizations, we will need to compare gene expression profiles of these CYP98 genes with genes known to be involved in lignin biosynthesis through analysis of large-scale gene expression data. These will also be mined to identify novel candidate genes involved in lignin biosynthesis in these lineages. Importantly, we need to establish approaches to generate mutants in these non-model species to functionally test a direct involvement of these genes in wood formation. We will generate loss-of-function mutants that will be characterized for changes typically found in flowering plant cyp98 mutants, including changes in lignin composition. Alterations in leaf morphology and metabolite composition will be assessed as these are affected in a non-lignin related moss cyp98 mutant. These experiments will show whether CYP98 is involved in lignin biosynthesis in ferns and conifers or if alternative pathways have evolved. If the latter holds true, candidate genes identified from the gene expression data will be tested for their ability to rescue the cyp98a3 loss of function phenotype in the model plant Arabidopsis and, if warranted, then further characterized biochemically and through reverse genetic approaches. Taken together, this program will test the evolutionary trajectory of lignin biosynthesis and may identify evolutionary optimizations of a biochemical pathway that was crucial for plants to conquer land and that defines wood properties and its uses by humans.